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Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica Cory Schwartz, Murtaza Shabbir-Hussain, Keith Frogue, Mark Blenner, and Ian Wheeldon ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00285 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 19, 2016

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ACS Synthetic Biology

Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica Cory Schwartz1, Murtaza Shabbir-Hussain2, Keith Frogue1, Mark Blenner2, and Ian Wheeldon1,* 1. Chemical and Environmental Engineering, University of California, Riverside 2. Chemical and Biomolecular Engineering, Clemson University * Corresponding Author. Email: [email protected] Abstract The yeast Yarrowia lipolytica is a promising microbial host due to its native capacity to produce lipid-based chemicals. Engineering stable production strains requires genomic integration of modified genes, avoiding episomal expression that requires specialized media to maintain selective pressures. Here, we develop a CRISPR-Cas9-based tool for targeted, markerless gene integration into the Y. lipolytica genome. A set of genomic loci was screened to identify sites that were accepting of gene integrations without impacting cell growth. Five sites were found to meet these criteria. Expression levels from a GFP expression cassette were consistent when inserted into AXP, XPR2, A08, and D17, with reduced expression from MFE1. The standardized tool is comprised of five pairs of plasmids (one homologous donor plasmid and a CRISPR-Cas9 expression plasmid), with each pair targeting gene integration into one of the characterized sites. To demonstrate the utility of the tool we rapidly engineered a semi-synthetic lycopene biosynthesis pathway by integrating four different genes at different loci. The capability to integrate multiple genes without the need for marker recovery and into sites with

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known expression levels will enable more rapid and reliable pathway engineering in Y. lipolytica.

Keywords: CRISPR-Cas9, genome editing, synthetic biology, metabolic engineering, standardized genetic tool, carotenoids


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A valuable microbial host for the biosynthesis of lipid-based chemicals is the oleaginous yeast Yarrowia lipolytica.1, 2 A set of gene overexpressions and knockouts has been identified that creates strains that accumulate lipids to more than 90% of cell dry weight (CDW).3-5 Synthetic and semi-synthetic pathways have also been designed including pathways for the biosynthesis of omega-3 fatty acids, pentane, carotenoids, and α-ketoglutarate among others.6-9 Its high capacity to produce and accumulate lipids has also made it a useful model for lipid metabolism.10, 11 In comparison to the model yeast Saccharomyces cerevisiae, Y. lipolytica has been more difficult to engineer. The high capacity of S. cerevisiae to undergo homologous recombination (HR) is the basis of many advanced synthetic biology and genetic engineering tools.12-14 HR in Y. lipolytica is significantly reduced and the number of advanced tools is relatively limited.15, 16 Integrative transformation and marker recovery by Cre-Lox recombination17 is available as are a number of stable expression plasmids,18 but genome integrations occur at low efficiency and have relied on selectable markers.19, 20 We recently added to the available genome engineering tools by developing a CRISPR-Cas9 system for use in Y. lipolytica,21 but the lack of advanced synthetic biology tools has limited rapid strain development and targeted markerless gene integration, which is favored in industry,22-24 is still challenging. Overexpression from chromosomal genes has been shown to be more consistent across cell populations than expression from episomal genes.25, 26 Targeted and random gene insertions into S. cerevisiae have also revealed that expression can vary upwards of 9-fold between genes integrated at different loci.27, 28 Similar effects have been observed in Escherichia coli and Bacillus subtilis, where gene expression has been shown to be higher from loci closer to the origin of replication.29, 30 The discovery of the type II CRISPR-Cas9 system from Streptococcus


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pyogenes and its adoption for genome editing has made less genetically tractable organisms more accessible and promises to enable more genome-wide and context-dependent expression studies.21, 31, 32 In this work, we developed a tool for markerless gene integrations into the Y. lipolytica genome at well-characterized sites. Using HR to repair CRISPR-Cas9 created double stranded breaks, we identified five loci where gene integration occurred at reasonably high efficiency (AXP, XPR2, A08, D17, and MFE1; Table 1). Experimental characterization of strains with an integrated model GFP expression cassette showed that disruption of the identified sites did not affect cell growth and that expression varied across selected sites. Finally, we demonstrated the utility of the gene integration tool by engineering a lycopene biosynthesis pathway in the Y. lipolytica genome without integrating selectable auxotrophic markers. To identify suitable genomic loci, we screened 17 unique sites for CRISPR-Cas9mediated HR of a heterologous humanized Renilla green fluorescent protein (hrGFP) expression cassette (Figure 1). The set of loci included four sites in cryptic sugar metabolism genes,20, 33 five sites in β-oxidation genes,7, 34 and five sites in annotated pseudogenes (Table S1).35 Three additional sites, AXP, XPR2, and LEU2, were screened as they are functionally disrupted in the PO1f strain. The β-oxidation genes (POX2, POX3, POX4, POX5, and MFE1) were selected because they are often disrupted when engineering high lipid accumulation,4, 7, 34, 36 the pseudogenes (A08, A11, B20, D17, and E07) were selected because they are annotated as not coding for functional protein products, and the cryptic sugar metabolism genes (XDH, XLK, XYR, and GAL10) were selected because they are not necessary for growth on glucose or lipid feedstocks.20, 33


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Table 1. Description of each site used as an integration target.

Figure 1. System for markerless gene integration in Y. lipolytica. (A) CRISPR-Cas9 expressing and homology donor plasmids. (B) Schematic representation of CRISPR-Cas9 induced homologous recombination. (C) Selected genomic loci with Y. lipolytica CLIB122 annotation and sgRNA targeting sequences. (D) Examples of hrGFP cassette integration into AXP including a schematic of PCR-based screening (top) and electrophoretic gel (bottom).

For each site, a homology donor plasmid was constructed with an hrGFP expression cassette flanked by 1 kb homology up and downstream of the targeted Cas9 cut site (Figure 1A, B). The donor plasmids were designed so that the targeted protospacer adjacent motif (PAM) was eliminated after recombination, thus avoiding Cas9 retargeting. Unique restriction sites between the homology regions were included for simple cloning with new genes of interest (Figure S1). This enables the generation of plasmids for integrating any gene of interest with


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only a single restriction digest and ligation. A second plasmid (pCRISPRyl) expressed codon optimized Cas9 from S. pyogenes and a targeting sgRNA.21, 37 sgRNAs sequences were designed using a previously defined scoring algorithm,38 with the highest scoring sgRNAs per gene selected for use. These sequences were designed to match the PO1f genome, which has high similarity but is not identical to the genomes of other Y. lipolytica strains. HR rates in Y. lipolytica using selectable genetic markers are reported to vary between 2 to 44%.15-17 Given the reported success of CRISPR-Cas9-mediated gene integration in S. cerevisiae and other yeasts,23, 39-41 we hypothesized that Cas9-induced double stranded breaks in the Y. lipolytica genome would increases HR rates and eliminate the need for integrated genetic markers. Figure 1D shows selected screening results of hrGFP integration into the AXP site. Gene integration was achieved by cotransforming pCRISPRyl and a HR donor plasmid specific to AXP. Two days of outgrowth in double selective media (LEU- and URA-) followed by plating on rich media produced isolated colonies. Screening for integrations was accomplished using a three-primer colony PCR. A 2 kb PCR product indicated the PO1f genotype, while a 1 kb PCR product resulted from the integration of the hrGFP expression cassette. In the selected examples shown in Figure 1D, three of six screened colonies showed successful integrations. Of the 17 tested loci, five resulted in efficient heterologous gene integration. MFE1 showed the highest efficiency at 69±25%. Integration into AXP occurred with an efficiency of 62±10%, while XPR2, A08, and D17 produced integrations with efficiencies of 48±13%, 53±33%, and 52±13% (Figure 2A, Table S2). Three additional sites (POX4, E07, and XDH) showed integrations with efficiencies less than 6%, while no integrations were observed in the other nine tested sites. It is possible that these sites were less amenable to gene integration due to DNA accessibility. Previous work has shown that chromatin structure and nucleosome


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occupancy influence the efficiency of CRISPR-Cas9-mediated processes in mammalian cells, this may also be the case in Y. lipolytica and other yeasts.42 In addition, no integrations were observed in control experiments with non-targeting sgRNAs and in the absence of the pCRISPRyl plasmid showing that the targeted Cas9 was necessary to induce integration (Figure 2A and Table S3). Disruption of non-homologous end-joining (NHEJ) through knockouts of KU70 and KU80 have previously been shown to increase HR efficiency in Y. lipolytica.15, 16 As such, we tested our system in a KU70 disrupted background. In the absence of NHEJ, integrations into LEU2, XLK, and XYR were possible, increasing HR efficiency from 0 to between 7 and 28% (Figure S2). In the case of the five high efficiency integration sites, KU70 disruption had little to no effect. In a previous work, it was found that disruption of KU70 resulted in an increase from 74% to 100% at a site within the MFE1 gene.21

Figure 2. Targeted genome integration sites in Y. lipolytica. (A) Homologous recombination (HR) efficiency of an hrGFP expression cassette into AXP, XPR2, A08, D17, and MFE1. Each site was tested


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with and without a targeting sgRNA. Integration rates are the average of three biological replicates with at least a total of 24 colonies screened across all replicates. (B) Growth rates of strains with hrGFP integrated into the indicated site. Growth curves are the average of three separate integrated colonies grown on rich media (YPD).

Colonies that showed positive integrations of hrGFP into AXP, XPR2, A08, D17, and MFE1 were cured of the two-plasmid system by overnight growth in rich media supplemented with 5-fluoroorotic acid (5-FOA). Plasmid removal was confirmed by an inability to grow in both URA- and LEU- medium (PO1f is an engineered URA and LEU auxotroph). Growth studies in rich media revealed that hrGFP integration into each of the high efficiency sites had no significant effect on growth (Figure 2B). The developed experimental protocol allows for the integration of a single gene in five days. The protocol includes two days of outgrowth in URA-/LEU- media after transformation, one day to produce colonies on solid rich media (for colony PCR-based screening), one day for plasmid removal in liquid culture, and one day to isolate colonies with successful integrations. A reduced time protocol of four days was also achieved by screening for gene integration after plasmid removal. In this case, after two days of outgrowth in URA-/LEU- selective liquid media a small volume of the liquid culture was used to inoculate rich liquid media containing 5-FOA to cure the plasmids. Plating on rich media produced isolated colonies that can be screened for successful integrations with pCRISPRyl and the HR donor plasmid already removed. Schematics of the protocols are shown in Figure S3. To quantify UAS1B8-TEF(136) driven expression of hrGFP, the integrated strains were analyzed by flow cytometry during mid-exponential and stationary phase (Figure 3A). For all five sites, expression was higher at stationary phase than during exponential phase (exponential


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to stationary phase effect, p

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